US 2016O23750 1A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2016/0237501 A1 SHARP et al. (43) Pub. Date: Aug. 18, 2016 (54) BIOMARKERS FOR DIAGNOSIS OF Related U.S. Application Data TRANSIENT SCHEMICATTACKS (62) Division of application No. 13/182,630, filed on Jul. (71) Applicant: The Regents of the University of 14, 2011, now abandoned. California, Oakland, CA (US) (60) Provisional application No. 61/364.334, filed on Jul. 14, 2010. (72) Inventors: Frank SHARP, Davis, CA (US); Xinhua ZHAN. Vacaville, CA (US); Publication Classification Glen C. JICKLING, Sacramento, CA (US): S. Claiborne JOHNSTON, San (51) Int. Cl. Francisco, CA (US) CI2O I/68 (2006.01) (52) U.S. Cl. (73) Assignee: The Regents of the University of CPC ........ CI2O 1688 (2013.0); CI2O 2600/158 California, Oakland, CA (US) (2013.01); C12O 2600/1 18 (2013.01) (57) ABSTRACT (21) Appl. No.: 15/043,577 The present invention provides methods and compositions for diagnosing and predicting the risk and cause of transient (22) Filed: Feb. 14, 2016 ischemic attacks (TIA). Patent Application Publication Aug. 18, 2016 Sheet 1 of 4 US 2016/0237SO1 A1 Standardized intensity s sis: iagnosis Controls xIA Figure IA-B Patent Application Publication Aug. 18, 2016 Sheet 2 of 4 US 2016/0237SO1 A1 & TA Cross-validated Probabilities (Thresholds 0.89) * Controls Controls TA ----------------------------------------------------------------------------------------------------------------------------------------- ... 0.9 O.8 O O 20 Subjects30 40 SO 50 Figure 2 Patent Application Publication Aug. 18, 2016 Sheet 3 of 4 US 2016/0237SO1 A1 Cross-validated Probabilities (Threshold=3.97) & TIA1 & A2 TIA1 T1A2 . .8 . 0. 4. O S O 20 25 Subjects Figure 3 Patent Application Publication Aug. 18, 2016 Sheet 4 of 4 US 2016/0237SO1 A1 FG. FG. A1 matrix metaliopeptidase 16 (MMP16) A2 matrix metaisopeptidase 16 (MMP16) 68. 8 8 p = 1.35E-7 p = 2.19E-7 g wis g 88: s 45: sess 456: s ge s esses xssess 8ss 888 sesses a: Controls A. As Controls A. A2 FG. FG. B1 matrix metallopeptidase 26 (MMP26) B2 atrix metallopeptile 26 (MMP26) 8.8; & siss s s e s s 2 as Controls A. As Contros A. A2 Figure 4A-B US 2016/02375O1 A1 Aug. 18, 2016 BOMARKERS FOR DIAGNOSIS OF present invention is based, in part, on gene expression profiles TRANSIENT SCHEMICATTACKS that provide insight into the immunological differences that exist in patients with TIAs. CROSS-REFERENCE TO RELATED APPLICATIONS BRIEF SUMMARY OF THE INVENTION 0001. This application is a divisional of U.S. application 0008. The present invention provides compositions and Ser. No. 13/182,630, filed on Jul. 14, 2011, which claims the methods for determining the occurrence, predicting the risk benefit of U.S. Provisional Application No. 61/364,334, filed of occurrence and predicting the cause of transient ischemic on Jul. 14, 2010, the entire disclosure of which is hereby attacks. incorporated herein by reference for all purposes. 0009. Accordingly, in one aspect, the invention provides methods for diagnosing a transientischemic attack (TIA) or a STATEMENT AS TO RIGHTS TO INVENTIONS predisposition for experiencing TIA, the method comprising: MADE UNDER FEDERALLY SPONSORED determining a level of expression of a plurality of TIA-asso RESEARCH AND DEVELOPMENT ciated biomarkers in a biological sample from a patient, wherein an increase or decrease of the level of expression 0002 This invention was made with Government support compared to a control indicates that the patient has suffered or under Grant No. NS056302, awarded by the National Insti is at risk of experiencing TIA, wherein the plurality of TIA tutes of Health. The government has certain rights in this associated biomarkers is selected from the biomarkers set invention. forth in Tables 1, 2, 5A, 5B, 5C, 5D, 7, 8 and 9. 0010. In some embodiments, the methods further com FIELD OF THE INVENTION prise obtaining a biological sample from the patient. In some 0003. The present invention provides methods and com embodiments, the biological sample is blood, serum or positions for diagnosing and predicting the risk and cause of plasma. transient ischemic attacks (TIA). 0011. In some embodiments, the determining step is per formed at 3 or fewer hours after a suspected ischemic event. BACKGROUND OF THE INVENTION In some embodiments, the determining step is performed at 3 or more hours after a suspected ischemic event, for example, 0004 Transient ischemic attacks (TIAS) are common, at about 6, 12, 24, 36, 48 or more hours after a suspected affecting over 300,000 persons per year in the United States ischemic event. In some embodiments, the determining step alone. Though TIA symptoms resolve by definition, TIAS are is performed at least 24 hours after a suspected ischemic far from benign. As many as 25% of TIA patients have recur event. rentischemic vascular events that occur within days to weeks 0012. In some embodiments, an increased expression following a TIA (1-3). Despite the high incidence and clinical level of one or more or all TIA-associated biomarkers importance, the development of therapies specifically tar selected from the group consisting of DKFZP434B061, geted toward TIA has been limited by the paucity of knowl FAM55D, FLJ30375, IGFBP5, LTER and SCN2A indicates edge regarding the underlying biology. Furthermore, the that the patient has suffered or is at risk of experiencing TIA. clinical diagnosis of TIA is imperfect and extensive evalua In some embodiments, an increased expression level of one or tion in those incorrectly diagnosed with TIA is costly (4). more or all TIA-associated biomarkers selected from the 0005 We have previously demonstrated that blood gene group consisting of GABRB2, ELAVL3, TWIST1, DPPA4, expression profiles in rats change following experimental DKFZP434P211, DLX6, ZNF479, ASTN2, SNX31, ischemic strokes and TIAS (5). Very brief focal ischemia in ALS2CR11, LOC440345 indicates that the patient has suf rats, simulating human TIA, elicits a dramatic change in brain fered or is at risk of experiencing TIA. tissue characterized by increased Heat Shock Protein 0013. In some embodiments, an increased expression (HSP70) expression, microglial activation and macrophage level of one or more or all TIA-associated biomarkers infiltration (6). This change in brain cellular function and selected from the group consisting of GABRB2, ELAVL3, inflammation alters blood immune cells, a process that can be COL1A1, SHOX2, GABRB2, TWIST1, DPPA4, detected using whole genome expression analysis (5). Fur DKFZP434P211, WIT1, SOX9, DLX6, ANXA3, EPHA3, thermore, the genes and associated functional pathways differ SOX11, SLC26A8, CCRL1, FREM2, STOX2, ZNF479, markedly between very brief focal ischemia and ischemic LOC338862, ASTN2, FOLH1, SNX31, KREMEN1, stroke (5). ZNF479, ALS2CR11, FIGN, RORB, LOC732096, GYPA, 0006 Human TIAS have also been associated with alter ALPL, LHX2, GALNT5, SRD5A2L2, GALNT14, OVOL2, ations in Systemic inflammation. TIA patients tend to have BMPR1B, UNC5B, ODZ2, ALPL, RASAL2, SHOX, elevated C-reactive protein (CRP) (7), IL-6, VCAM-1 and C19orf59, ZNF114, SRGAP1, ELAVL2, NCRNA00032, cytokine levels, as well as elevated leukocyte counts (8-10). LOC440345, FLJ30375, TFPI, PTGR1, ROBO1, NR2F2, Lp-PLA2, a marker of unstable atherosclerotic plaque, is also GRM5, LUM, FLJ39051, COL1A2, CASP5, OPCML, associated with TIA (11-12) as are fibrinogen (13-14) and TTC6, TFAP2B, CRISP2, SOX11, ANKRD30B, FLJ39051, D-Dimer (15). Whether such biological differences represent SCN2A, MYNN, FOXA2, DKFZP434B061, LOC645323, a cause or consequence of TIA remains unclear. However, SNIP, LOC645323, LOC374491, ADAM30, SIX3, better understanding of the pathophysiology represented by FLJ36144, CARD8, KREMEN1, RP1-127L4.6, FAM149A such differences will facilitate development of treatments B3GAT2, SPOCK3, G30, ITGBL1, IQGAP3, C7orfA5, targeted to TIA. ZNF608, LOC375010, LRP2, TGFB2, SHOX2, HOXC4/// 0007 Gene expression has been useful for identifying dif HOXC6, ELTD1, FAM182B///RP13-401N8.2, PROO478, ferences between patients with ischemic stroke and controls LIFR, FOLH1, EHF, NDST3, BRUNOL5, LOC728460, (16-18), but such studies have not been applied to TIA. The PDE1A, POU2AF1, FAT1, PCDH11X///PCDH11Y, US 2016/02375O1 A1 Aug. 18, 2016 FLJ37786, SLC22A4, DHRS13, EHF, MEG3, PIWIL1, MMP 19, MMP26, COL1A1, COL1A2, COL3A1, LOC203274, LOC100.133920///LOC286297, DMRT1, COL10A1, COL11A1, COL25A1, COL27A1, FGFs and ADM, VWA3B, GAFA3, HESX1, ADAMDEC1, CAV1, EGFR is increased in comparison to the control, and the LAMB4, TPTE, PPP1R1C, HPSE, AIM2, RUNDC3B, patient is determined to have atherosclerosis. CARD16, FAM124A, MGC39584, OSM, RFX2, MYBPC1, 0019. In some embodiments, the patient is exhibiting LTBR, C18orf2, SNRPN, FLJ36031, IL1B, TRPM1, symptoms of TIA. In some embodiments, the patient is OSTCL, MAPK14, KCNJ15///LOC100131955, FIGN, asymptomatic. HNT, S100A12, CHIT1, C7orf53, FAM13A1, GNAO1, 0020. In some embodiments, the methods further com MAPK14, FAM55D, PRKD2, LIMK2, C18orf54, IGFBP5, prise the step of providing an appropriate treatment or pre EVI1, PLSCR1, FOXC1, LOC646627, ZNF462, CNTLN, vention regime for TIA to the patient. ZNF438, DEFB105A///DEFB105B, LOC34.0017, C1orf67, 0021. In some embodiments, the level of expression of the ACSL1, ADH1B, SLC2A14///SLC2A3, IL1B, ST3GAL4, biomarker is determined at the transcriptional level. For UBE2J1, PNPLA3 and PAPPA indicates that the patient has example, RNA levels of the biomarker can be determined. suffered or is at risk of experiencing TIA. The RNA can be mRNA, rRNA, tRNA or microRNA 0014. In some embodiments, a decreased expression level (miRNA). In some embodiments, the level of RNA expres of one or more or all TIA-associated biomarkers selected sion is determined using a microarray. from the group consisting of ATG9B, DIP2C, EDAR, 0022. In some embodiments, the level of expression is GSTM1, GUSBL2, SMURF2, ZNF512B indicates that the determined by detecting hybridization of an TIA-associated patient has suffered or is at risk of experiencing TIA. gene probe to gene transcripts of the biomarkers in the bio 0015. In some embodiments, a decreased expression level logical sample. of one or more or all TIA-associated biomarkers selected 0023.